Developmental Cell

Supplemental Information

Migration of Founder Epithelial Cells Drives

Proper Molar Tooth Positioning and Morphogenesis

Jan Prochazka, Michaela Prochazkova, Wen Du, Frantisek Spoutil, Jolana Tureckova,

Renee Hoch, Tomomi Shimogori, Radislav Sedlacek, John L. Rubenstein, Torsten

Wittmann, and Ophir D. Klein

Supplemental Figures:

Supplemental Figure 1: A narrow window of Fgf8 expression labels the future tooth

germ and is distinct from the site of Shh expression (related to Figure 1). (A-F) Lineage

tracing experiments using constitutive Fgf8ires-cre;R26RLacZ provided similar results to

experiments in which Fgf8creER was induced at approximately E11.5 (1 day after tamoxifen

injection) shown in Figure 1. The constitutive cre activity shown here provided higher

efficiency of recombination that was more suitable for live imaging experiments and

statistical analysis. (G-I) Induction of cre in Fgf8creER;R26mT/mG embryos by tamoxifen

injection at E10.75 showed exclusive labeling of the oral epithelium without any labeling in

adjacent mesenchyme at E11.5 (G), E12.5 (H) and E14.5 (G). (J-L) Induction of cre in

Fgf8creER;R26RLacZ embryos by tamoxifen injection at E11.5 (J) showed only a few labeled

cells in the E14.5 tooth germ. Injection of tamoxifen at E12.5 (K) or E13.5 (L) showed few to

no labeled cells. Asterisk labels the site where jaw joint was cut. (M-R) Comparison of clonal

growth of dental epithelium (N-P) and oral epithelium of tongue (Q). (R) Statistical plot of

clone probability density between tooth and tongue epithelia shows highly significant

difference in clonal behavior of dental and tongue epithelium. Mann-Whitney unpaired, non-

parametric test; p = 1.5x10-10. (S-Z) ShhEGFP;Fgf8LacZ co-localization during embryonic

development at E11.5 (T-V, 50 mg wet weight) and E12.5 (X-Z, 100 mg wet weight).

Supplemental Figure 2: The descendants of Fgf8-expressing cells are organized in a

transient posterior rosette structure that is sensitive to fixation (related to Figure 2). (A)

Quantification of cell shapes from Figure 2C, D shows that cells lost their elongated shape

after fixation in 4% PFA. (B, C) Images from Fgf8ires-cre;R26RConfetti embryos of large rosette

cells after fixation (B) and live (C). (D, E) Higher magnification view (400x) of central part of

large rosette shows enrichment of E-cadherin. (D) EcadCFP, (E) EcadCFP;R26RTomato. (F)

Similar view with use of LifeAct embryo showing actin enriched rosette structure

(arrowhead).

Supplemental Figure 3: The descendants of Fgf8-expressing cells are organized in a

transient posterior rosette structure that is released by intraepithelial migration during

development (related to Figure 2). (A-C) Automatic cell tracking in rosette. (A)

Segmentation of the posterior mandible into rosette population (blue) and surrounding cells

(yellow). (B) Tracks and displacement vectors for all cells. (C) Quantification of cell

migration parameters for both populations. Plots are presented with end of whiskers set at the

1.5x interquartile range above the third quartile and below the first quartile; open circles mark

maximal outliers. Student T-test, with * = p<0.05; ** = p<0.01; *** = p<0.0001. (D-F)

Automatic cell tracking in posterior mandible with use of membrane-targeted GFP

(R26mT/mG). (D) Automatically recognized cells and tracks. (E) Color coded tracks. (F) Color

coded tracks merged with displacement vectors. (G-W) Effect of blebbistatin treatment on

tooth morphogenesis. (G) Cartoon showing region studied. (H, J) K14-GFP visualization of

oral epithelium in control mandible culture (H) and blebbistatin treated mandible (J).

Morphology of dental epithelium was 3D reconstructed from optical confocal sections (I, K).

Blebbistatin treated mandible (K) shows mislocalised shallow circular invagination instead of

dental lamina formation as in control (I). (L-W) Dysregulation of expression of genes after

blebbistatin treatment: Pitx2 in dental epithelium (L, R); Msx1 in dental mesenchyme (M, S);

Shh in enamel knot (N, T); Eda in dental epithelium (O, U); Wnt10b in enamel knot (P, V);

Bmp4 in enamel knot and dental mesenchyme (Q, W). Scale bars: 100 µm.

Supplemental Figure 4: Directed cell migration occurs specifically in progeny of Fgf8-

expressing cells from the rosette (related to Figure 2). (A) The mandible was segmented

into four parts (blue – rosette, green – incisor, purple – labial, red – posterior areas). All

descendants of Fgf8-expressing cells were automatically tracked (B) and average values of

cell migration parameters (track length and displacement) were plotted (C). (D-G)

Visualization of track length and displacement for cells in each part of mandible (D) in

rosette, (E) in incisor, (F) in labial, (G) in posterior. (H-J) The specificity of cell migration

properties was tested with additional cre drivers to determine if the origin of cells is important

for the migratory behavior. Sox2creER was used to label an additional epithelial population

within the embryonic mandible, and cells were automatically tracked (I). Tracks of Sox2-

derived cells showed significantly lower migration track length, displacement and straightness

in movement than Fgf8 derived cells (J). (K-M) Live imaging of mesenchymal cells adjacent

to dental epithelium. Cells show only limited movement (L), which is in a circular pattern in

the presumptive rosette area (M represents white rectangle in L). (N-P) Quantification of cell

migration parameters: (N) track length, (O) displacement, (P) straightness among cells from

different segments of the mandible (color coded), as well as Sox2 progeny and mesenchymal

cells labeled with Wnt1cre. Plots are presented with end of whiskers set at the 1.5x

interquartile range above the third quartile and below the first quartile; open circles mark

maximal outliers. Student T-test, with * = p<0.05; ** = p<0.01; *** = p<0.0001. (Q) Track

translation analysis to compare the general direction of movement of descendants of Fgf8-

expressing cells from rosette with Sox2 progeny (see also Supplemental Video 3).

Supplemental Figure 5: Descendants of Fgf8-expressing cells have features of collective

migration, can form supernumerary tooth in the diastema, and are attracted by SHH

(related to Figures 2-5). (A-D) Descendants of Fgf8-expressing cells maintain E-cadherin

expression; quantification of fluorescence intensity between neighboring cells (D) suggests

that the cell contacts between descendants of Fgf8 expressing population are enriched in E-

cadherin. X-axis in D represents fluorescence units. (E-J) Whole mandible view showing

lower magnification versions of images in Figure 3D-H, as well as of Wnt10b. (K-U)

Proliferation and apoptosis assay on cyclopamine and SU5402 treated organ cultures. (K-M)

control, (O-Q) cyclopamine, (Q-S) SU5402. (T) Quantification showing no significant

differences in proliferation in prospective dental epithelium. (U) Quantification of apoptosis

shows no significant differences in cell death within cultured dental epithelia. (V-Y) Spry4

null embryonic molars at E14.5. Compared to controls (V), in Spry4 null mice destined for a

supernumerary tooth phenotype (W), the descendants of Fgf8-expressing cells expanded

anteriorly and were localized in the supernumerary tooth primordium. (X) In Spry4 null

embryos without a supernumerary cap, descendants of Fgf8-expressing cells lineage were not

evident in the diastema. S – supernumerary cap, M1 – first molar primordium. (Y)

Quantification of the expansion of descendants from Fgf8-expressing cells along the antero-

posterior length of the dental epithelium. (Z-Zd) Descendants of Fgf8-expressing cells are

more attracted to SHH-soaked beads than control beads. Standard deviation was used for error

estimates, Student T-test * = p<0.05; ** = p<0.01; *** = p<0.0001.

Supplemental Table:

Supplemental Table 1 related to quantification of data from Figure 1, Figure 3, Figure 4, Figure 5, Supplemental Figure 3, Supplemental Figure 4 and Supplemental Figure 5. Outline of table is provided below; actual table provided as .xls sheet.

Sheet name Refers to Description

Fig1_Supplemental data Figure 1w-z; Supplemental Figure 2n,o Statistical evaluation of clonal growth

Fig3_Supplemental data Figure 3p-r Formation of dental lamina after Shh and Fgf inhibition

Fig4a_Supplemental data Figure 4a-h, q Formation of dental lamina in conditional cell autonomous mutants

Fig4b_Supplemental data Figure 4i-l, r Epithelial cell migration in conditional cell autonomous mutants

Fig5_Supplemental data Figure 5 Epithelial cell migration after Shh annd Fgf inhibition

SFig3_Supplemental data Supplemental Figure 3c-e Automatic cell tracking data in rosette

SFig4a_Supplemental data Supplemental Figure 4a-g, n-p Automatic cell tracking data in rosette and other parts of mandible

SFig4b_Supplemental data Supplemental Figure 4h-j, n-p Automatic cell tracking of Sox2-positive cells

SFig4c_Supplemental data Supplemental Figure 4k-m, n-p Automatic cell tracking data in mesenchyme

SFig5a_Supplemental data Supplemental Figure 5d-g Length of dental lamina in Spry4 null embryos with and without supernumerary tooth germ

SFig5b_Supplemental data Supplemental Figure 5h-l Cell attraction to SHH soaked beads and EcadCFP intensity

Supplemental Video Legends:

Supplemental Video 1 related to Figure 1: 3D reconstruction generated in Imaris from Scale cleared K14-GFP-actin control and Fgf8creER;R26RDTA embryo.

Supplemental Video 2 related to Figure 2: (a) 14 hour time-lapse imaging of rosette

structure in Fgf8ires-cre;R26RConfetti embryonic mandible showing rosette release followed by

oriented cell movement. (b) 30 hour time-lapse imaging of control embryonic mandible in

Fgf8ires-cre;R26RConfetti embryo (YFP channel) showing cell migration within oral epithelium.

Most of the cell movement originates in the rosette region, and all cell movement is oriented

towards the dental lamina site. (c, d) Higher magnification time-lapse imaging of individual

cell clusters from Fgf8 expressing area show membrane dynamics and protrusion formation

during migration. (e) 48 hour time-lapse imaging of embryonic mandible in Fgf8ires-

cre;R26mT/mG embryo showing cell migration within oral epithelium. Most of the cell

movement originates in the rosette region, and all cell movement is oriented towards the

dental lamina site, where a strong contraction is visible. Asterisk indicates center of the

rosette.

Supplemental Video 3 related to Figure 2: 14 hour time-lapse imaging of embryonic

mandible in Sox2creER;R26mT/mG embryonic mandible showing non-directed, non-oriented cell

movement.

Supplemental Video 4 related to Figure 4 and Figure 5: 36 hour time-lapse imaging of

embryonic mandible showing cell migration within oral epithelium in (a) Fgf8creER;R26mT/mG

control embryo, (b) Fgf8creER/flox;R26mT/mG, (c) Fgf8creER;R26mT/mG;Smoflox/flox and (d)

Fgf8creER;R26mT/mG; R26RSmoM2 embryos. (e) 30 hour time-lapse imaging in YFP channel of

Fgf8ires-cre;R26RConfetti embryo after cyclopamine and SU5402 treatment. (f) Cells from

cyclopamine treated mandible show a disruption in oriented cell migration within the oral

epithelium. Cells from SU5402 treated mandible show a decrease of cell migration, with most

cells remaining in the rosette region. Asterisk indicates center of the rosette.

Supplemental Material and Methods:

Mouse lines

The following transgenic mouse strains were used: Fgf8LacZ (MGI: 3612999) (Grieshammer et

al., 2005), Fgf8ires-cre (MGI: 4839641) (Toyoda et al., 2010), R26RLacZ (MGI: 1861932)

(Soriano, 1999), R26mT/mG (MGI: 3716464) (Muzumdar et al., 2007), R26RConfetti (MGI:

4835542) (Snippert et al., 2010), R26RDTA (MGI: 3618991) (Wu et al., 2006), K14EGFP/Actb

(K14-GFP-Actin) (MGI: 4421514) (Vaezi et al., 2002), Ptc1LacZ (MGI: 1857447) (Goodrich

et al., 1997), Gli1LacZ (MGI: 2449767) (Bai et al., 2002), Spry4– (MGI: 3701941) (Klein et al.,

2006), R26RRFP (MGI: 3809524) (Madisen et al., 2010), ShhEGFP/cre (MGI: 3053959) (Harfe et

al., 2004), Smoflox (MGI: 2176256) (Long et al., 2001), R26RSmoM2 (MGI: 3576373) (Jeong et

al., 2004), Fgf8flox (MGI: 2150347) (Meyers et al., 1998), EcadCFP (MGI: 4838590) (Snippert

et al., 2010), Fgf8creER (Hoch et al., 2015) Wnt1cre (Danielian et al., 1998) (MGI: 2386570)

Sox2creER (Arnold et al., 2011) (MGI: 5295990), LifeAct (Riedl et al., 2010) (MGI: 4831038).

Tamoxifen was administrated to pregnant females intraperitoneally at a dose of 9 mg per 40 g

of mouse weight. All embryos were staged by embryonic day and wet body weight to

optimize comparison between litters. Representative body weights for individual stages were

as follows: E11.5 – 50 mg, E12.5 – 90-100 mg, E14.5 – 230-280 mg.

Mating and embryo harvest schemes

male female embryos harvested

Fgf8LacZ B6 wild-type (WT) Fgf8LacZ

Fgf8ires-cre R26RLacZ Fgf8ires-cre;R26RLacZ

Fgf8ires-cre R26RConfetti Fgf8ires-cre ;R26RConfetti

Fgf8ires-cre R26mT/mG Fgf8ires-cre;R26mT/mG

ShhEGFP/cre R26RConfetti ShhEGFP/cre;R26RConfetti

Fgf8creER;K14EGFP/Actb R26RDTA Fgf8creER;K14EGFP/Actb;R26RDTA Fgf8creER;K14EGFP/Actb

Fgf8creER R26RLacZ Fgf8creER;R26RLacZ

Fgf8creER R26mT/mG Fgf8creER;R26mT/mG

Fgf8LacZ;ShhEGFP/cre B6 WT Fgf8LacZ;ShhEGFP/cre

Fgf8creER R26RRFP;EcadCFP Fgf8creER;R26RRFP;EcadCFP

LifeAct B6 WT LifeAct

Fgf8creER;K14EGFP/Actb Fgf8flox Fgf8creER/flox;K14EGFP/Actb Fgf8creER;K14EGFP/Actb Fgf8creER;K14EGFP/Actb; Smoflox/+ Smoflox/flox Fgf8creER;K14EGFP/Actb;Smoflox/flox Fgf8creER ;K14EGFP/Actb;

Smoflox/+

Fgf8creER;K14EGFP/Actb R26RSmoM2 Fgf8creER;K14EGFP/Actb;R26RSmoM2 Fgf8creER;K14EGFP/Actb Spry4+/-; Fgf8creER;R26mT/mG Spry4+/-;R26mT/mG Spry4-/-;Fgf8creER;R26mT/mG Spry4+/-;

Fgf8creER;R26mT/mG Spry4+/+;Fgf8creER; R26mT/mG

Gli1LacZ B6 WT Gli1LacZ

Ptc1LacZ B6 WT Ptc1LacZ

Sox2creER R26mT/mG Sox2creER;R26mT/mG

Wnt1cre R26RConfetti Wnt1cre;R26RConfetti

Histology and in situ hybridization

For histological analysis of LacZ expression, samples were fixed in Mirsky’s fixative

(National Diagnostics) overnight and subsequently stained in X-Gal solution. Stained samples

were post-fixed in 4% PFA and imaged on a Leica MZ 16F stereoscope with Spot 2.3.1

camera (Diagnostic Instruments). Samples were embedded in paraffin, cut in 10 µm sections,

and counter-stained with nuclear fast red (Sigma). Imaging of histological sections was done

with a Leica DM5000 microscope. Three embryos at each stage were analyzed. To analyze

3D morphology of dental epithelium, samples were fixed for 1 hour in 4% PFA and then

cleared in scaleA2 solution for two weeks as described in (Hama et al., 2011). Imaging was

done with an inverted Leica SP5 LSM, optical sections were generated every 4 µm, and 3D

reconstructions were obtained using Imaris software (Bitplane) A minimum of 4 samples was

processed for 3D analysis and quantification of each stage and genotype. In situ hybridization

was done according to standard protocols. To generate digoxigenin labeled riboprobes,

plasmids containing Shh, Fgfr2, Etv4, Bmp4, Wnt10b, Eda, Pitx2 and Msx1 sequences were

used for in vitro transcription. A minimum of 3 independent samples was processed for in situ

hybridization. Cell proliferation was analyzed by adding an EdU pulse into organ cultures (10

µM final concentration) (Life technologies) and co-staining with TUNEL kit (Roche)

according to manufacture protocol.

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